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Chitosan-Cu-salen/Carbon Nano-Composite Based Electrode for the Enzyme-less Electrochemical Sensing of Hydrogen Peroxide
Harishchandra Digambar Jirimali1,2*, Duraisamy Saravanakumar1,3, and Woonsup Shin1*
1Department of Chemistry and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul 121-742 Republic of Korea
2School of Chemical Sciences, North Maharashtra University, Jalgaon (MS) 425001 India
3Department of Chemistry, School of Advanced Sciences, VIT University, Vellore-632014, India
ABSTRACT
Cu-Salen complex was prepared and attached into chitosan (Cs) polymer backbone. Nanocomposite of the synthesized polymer was prepared with functionalized carbon nano-particles (Cs-Cu-sal/C) to modify the electrode surface. The surface morphology of (Cs-Cu-sal/C) nanocomposite film showed a homogeneous distribution of carbon nanoparticles within the polymeric matrix. The cyclic voltammogram of the modified electrode exhibited a redox behavior at -0.1 V vs. Ag/AgCl (3 M KCl) in 0.1 M PB (pH 7) and showed an excellent hydrogen peroxide reduction activity. The Cs-Cu-sal/C electrode displays a linear response from 5×10-6 to 5×10-4 M, with a correlation coefficient of 0.993 and detection limit of 0.9μM (at S/N = 3). The sensitivity of the electrode was found to be 0.356μAμM-1cm-2.
Keywords : Electrochemical sensor, Hydrogen peroxide, Chitosan, Copper complex, Polymer nano-composite Received : 10 November 2017, Accepted : 21 April 2018
1. Introduction
Electrode modifications with redox-active moieties or a functional groups has gain a great interest, due to their fundamental importance in electrochemical catalysis and bio-sensing where transition metal com- plexes are used as an electro-catalyst due to their tun- able redox potential [1-3]. Tetra dentate Schiff base ligands have been extensively used as catalysts in organic reactions and bio-mimetic enzymatic reac- tions [4,5]. Salen based electro-active complexes and their polymers have potential applications in the field of electro-catalysts, chemical sensors and optical devices [6,7]. The metal complexes are often coated on the electrode surfaces either by electro-deposition or immobilizing them with polymer matrix for the catalytic purposes[8-10]. Copper complexes of salen- type ligands are important in some biochemical pro-
cesses, and DNA cleavage and antimicrobial activity [11-14]. Cu-salen complexes and metal organic frame work have been prepared and their properties had been studied well [15-17].
Chitosan is an amino-polysaccharide prepared by deacetylation of chitin, which is discarded as indus- trial waste so it can be considered as a low cost and nonhazardous polymer [18,19]. Chitosan bears free amine groups which are used for the immobilization of the metal complexes and redox mediators [20-23].
Currently, much attention has been focused on devel- oping new nano-materials, which are used for signal amplification in electrochemical methods [24]. Con- ducting nanomaterials with a larger surface area are the potential candidates for biomolecules immobili- zation and signal amplifications [25]. Among the available materials used, carbon nano-materials are the most exciting because of their unique physical, chemical and mechanical properties [26,27].
Hydrogen peroxide is a vital compound in the bio- logical transformations also widely used in several
*E-mail address: shinws@sogang.ac.kr, hdj739@gmail.com DOI: https://doi.org/10.5229/JECST.2018.9.3.169
Journal of Electrochemical Science and Technology
Recently, the synthesis of a new bimetallic metallo- porphyrinic framework of Cu(II) complex and its electrochemical application for the hydrogen perox- ide detection has been reported [34].
The present work describes the synthesis of Cu- salen coordinated chitosan polymer (Cs-Cu-sal).
Redox active nano-composite of Cs-Cu-sal was pre- pared by simple mixing with functionalized carbon nanoparticles. Cs-Cu-sal/C composite was drop coated on the printed carbon electrodes and these modified electrodes were tested for H2O2 sensing by cyclic voltammetric and i-t curve techniques. The Cs- Cu-sal/C electrode showed the sensitivity of 0.356μA μM-1cm-2 with the linear range of 5×10-6 to 5×10-4 M for the hydrogen peroxide sensing.
2. Experimental
2.1 Reagents
Chitosan (lower mol. wt.), copper(II)acetate mono- hydrate, ethylene diamine, 2-hydroxy benzaldehyde, acetic acid and hydrogen peroxide were obtained from Sigma Aldrich and membrane filters from the Millipore. All the chemicals were of analytical grades and used without further purification. Water purified from Millipore system was used in all the experiments. 0.1 M phosphate buffer (pH 7.0) solu- tion was prepared with Na2HPO4 and NaH2PO4.
2.2 Surface characterizations
The surface morphology of the composite film on a printed carbon electrode was recorded with the use of a field emission scanning electron microscope, Hita- chi S-4300 at 15 KV.
2.3 Electrochemical experiments
Cyclic voltammetry was performed with printed carbon electrode (PCE, 5 mm, SE100, Zensar R &
(244 mg) in 1:2 molar ratio were added in ethanol and the mixture was heated to reflux for 1 h. Bright yellow crystalline solid of salen [N,N-ethylen- ebis(salicylidene-aminato)] ligand was formed which was collected by filtration and washed thoroughly with ethanol.
2.4.2 Neat Cu-salen complex
Solution of Cu(CH3COOH) (181 mg) in 10 mL (methanol:water; 7:3 v/v) was added slowly to salen (272 mg) in 15 mL methanol under stirring at room temperature. The resulting mixture was heated to reflux at 80oC for 1 h and the solid complex formed was isolated by filtration. The dark green crystals of Cu-salen complex thoroughly washed with methanol and dried, which is referred as Cu (II)-salen.
2.4.3 Cs-Cu-sal polymer
Neat Cu-salen solution in methanol was added dropwise to 1% chitosan in 1% aqueous acetic acid and stirred overnight. The resulting Cs-Cu-sal was filtered through 10,000 molecular weight cut-off polycarbonate membrane to remove the low molecu- lar weight polymer. The obtained polymer was pre- cipitated with acetone, collected and dried under the vacuum.
2.5 Treatment of carbon
Commercially available carbon black (C) powder was treated with acid as per earlier reported proce- dure [20]. The carbon black powder was boiled in 5 M HNO3for a shorter time, filtered and repeatedly washed with deionized water. Functionalized carbon black was dried and used for the Cs-Cu-sal polymer nano-composite preparation.
2.6 Preparation of electrodes
200μL of 1% Cs-Cu-sal polymer in 1% acetic acid
(10 mg/mL) and 2 mg of acid treated carbon (C) powder were mixed and sonicated for 1 minute. 2μL of this Cs-Cu-sal/C composite material was drop coated on the PCE. Air dried electrodes were washed with water and used for the further electrochemical and other characterization.
3. Results and Discussion
Cs-Cu-sal polymer was synthesized in two steps procedure as presented in Scheme 1. In the first step, Cu-salen complex was prepared by mixing salen ligand and Cu acetate in methanol : water solvent.
The chitosan polymer was modified with Cu-Sal complex by simple mixing of 1% chitosan in 1% ace- tic acid solution with the metal complex. The com- posite of the Cu complex attached polymer with functionalized carbon nano powder was prepared by mixing them and sonication. The PCEs were modi- fied by drop coating of Cs-Cu-sal/C and dried it at room temperature.
Fig. 1(A) showed the SEM image of Cs-Cu-sal/C polymer composite film and Fig. 1(B) showed the image of the pristine carbon nanoparticles. The sizes of the pristine carbon particles and the carbon parti- cles embedded in the polymer composite were observed in the range of 50 to 100 nm. The function- alized carbon particles having slightly negative charge and Cs-Cu-sal polymer having slightly posi- tive charge in acidic pH due to the protonation of the free amines of the chitosan polymer. The polymer composite was prepared by sonication of functional- ized carbon and polymer in the solution, the polymer with positive charge can form the thin film on the negatively charge high surface area carbon particles.
The homogeneous distribution of the carbon particles in the polymer produces a three dimensional homo- geneous porous film and the presence of polymer was in the composite was supported by the electro- chemical characterizations.
Fig. 1. SEM images of Cs-Cu-sal/C modified PCE surface view (A) and pristine carbon particles (B).
Scheme 1. Synthesis of Cs-Cu-sal polymer.
3.1 Electrochemical characterization of Cs-Cu-sal/C The cyclic voltammogram of Cs-Cu-sal/C elec- trode was recorded in 0.1M phosphate buffer (pH 7) under Ar, shown in Fig. 2(A). The modified elec- trode exhibited a set of redox peaks at 0.15 V and - 0.17 V at the scan rate of 50 mV/s due to Cu(II) to Cu(I) couple [35]. The overlay of the cyclic voltam- mograms of Cs-Cu-sal/C film at scan rate of 10 mV/
s to 200 mV/s indicated that the redox peak current and peak to peak separation were linearly increased with the increased in scan rate. Fig. 2(B) showed the linear dependence of the redox peak current to the scan rate, suggested that the charge transport through the film occurs under the surface confined phenomenon of electron transfer at Cs-Cu-sal/C electrode. Similar behavior was reported by Van Bui , for the ferrocene brushes modified electrodes [36].
This newly prepared Cs-Cu-sal/C film showed ade-
quate stability in the aqueous solutions as the cur- rent of the peaks are not decreased on continuous cycles.
3.2 Electrocatalytic effect on reduction of hydrogen peroxide
The usefulness of the Cs-Cu-sal modified electrode for H2O2 sensing was further demonstrated by cyclic voltammetry. Cyclic voltammograms of the modi- fied electrode were recorded in 0.1 M PB at pH 7 in the presence and absence of 5 mM H2O2 at the scan rate of 10 mV/sec (Fig. 3) under Ar. The current of the reduction peak of the Cu (II) to Cu (I) redox cou- ple at -200 mV is increased in the presence of 5 mM H2O2 (dashed line) indicating the hydrogen peroxide reduction. The reduction of the hydrogen peroxide and the possible reaction mechanism is proposed as follows.
Cu(II)-sal + e-→ Cu(I)-sal (1) 2Cu(I)-sal + H2O2 + 2H+ → 2 Cu(II)-sal + 2H2O (2)
3.3 Effect of scan rate and pH
Effect of scan rates (ν) on the sensing of H2O2 in terms of catalytic currents was investigated in the range of 10 mV/s to 80 mV/s. Fig. 4(A) shows the CVs of the Cs-Cu-sal/C at different scan rates in the potential window of -0.6 to +0.5 V in PBS (pH 7.0).
From Fig. 4(B), it can be observed that the cathodic peak currents varied linearly with the increase in scan rate and this indicates the surface confined electron Fig. 2. Cyclic voltammograms of the Cs-Cu-salen/C
composite electrode in 0.1 M phosphate buffer at the scan rates of 20 mV/sec to 200 mV/sec (A) and plot of scan rate vs. current (B).
Fig. 3. Cyclic voltammograms of Cs-Cu-sal/C modified PCE in 0.1 M phosphate buffer (solid line) and phosphate buffer with 5 mM H2O2 (dash line) at 10 mV/sec.
transfer at the Cs-Cu-sal/C modified electrode sur- face. The effect of pH on the H2O2 reduction also checked by CV in the PB with pH ranges from 5 to 9 (Fig. 5(A)). Hydrogen peroxide reduction potential was slightly shifted with pH, while decreased in cur- rent was observed from pH 5 to pH 9 (Fig. 5(B)). The H2O2 reduction is the H+ dependent process accord- ingly there is shifting of the potential occurred as th pH of the phosphate buffer changes from pH 5 to pH 9 [37]. The modified electrode did not show signifi- cant change in the catalytic response when the pH was varied from 5 to 9 (variation in current response is within 5μA). Though the modified electrode was stable in the investigated pH range, pH 7 was chosen as the working pH for further experiments as neutral pH is preferable for analyzing H2O2 in biological samples.
3.4 Amperometric H2O2 detection
The amperometric hydrogen peroxide sensing was studied by typical current-time response of the bio- sensor for successive additions of H2O2 in the contin- uously stirred PB at an applied potential of -0.3 V under the optimized experimental conditions (Fig. 6 (A)). The rapid H2O2 response was achieved when the H2O2 was added in the stirred phosphate buffer solution and achieved 95% of steady-current in 5 seconds. The plot of the hydrogen peroxide concentration vs. ampero- metric current (Fig. 6(B)) showed the linear range of hydrogen peroxide concentration from 5× 10-6M to 5× 10-4 M and the detection limit of 0.9 mM with the sensitivity of 0.356 mA mM-1cm-2. The present sen- sor exhibits a comparable detection limit, linear detec- tion range and higher sensitivity than the recently reported electrodes for the detection of hydrogen per- oxide in neutral aqueous solutions [34,35,38].
Fig. 4. Cyclic voltammograms of Cs-Cu-sal/C modified PCE in 0.1 M phosphate buffer containing 1 mM H2O2 at the scan rate of 10, 20, 30, 40, 50, 60, 70, 80 mV/sec from inner to outer (A), and the cathodic peak current vs. scan rate (B).
Fig. 5. Cyclic voltammograms of Cs-Cu-sal/C modified PCE in 0.1 M phosphate buffer containing 1 mM H2O2 in pH 5 (red), 6 (green), 7 (black), 8 (blue), and 9 (cyan blue) respectively (A) and plot of cathodic peak current vs. pH (B).
3.5 Effect of interferences and stability
It is known that the nitrate and ascorbic acid can be electrochemically reduced at the negative potential as of hydrogen peroxide and considered as an interfer- ence in the hydrogen peroxide sensing [38,39]. The effect of the interfering nitrate and ascorbic acid was examined by amperometric analysis. Fig. 7 shows the amperometric i-t curve at Cs-Cu-sal/C modified elec- trode in stirred phosphate buffer. Addition of 0.5 mM of H2O2 showed instant increase in the current (a) and followed by the addition equimolar ascorbic acid (b) and nitrate (c) solutions didn’t cause any effect on H2O2 peak current. The stability of the modified elec- trode was tested by subjecting to potential cycling in the presence and absence of H2O2. The modified electrode shown 5% relative decrease in peak current after 50 cycles in the absence of H2O2, while it exhib- ited 7.4% decrease in current response in the pres-
ence of H2O2. The stability data indicates the usefulness of the present electrode in the detection of hydrogen peroxide.
4. Conclusions
Cs-Cu-sal polymer and it’s composite with carbon nanoparticles were prepared. The composite film on the PCE electrodes was prepared by drop coating method. The modified electrodes was characterized and tested for the hydrogen peroxide sensing in neu- tral aqueous solutions. Method of sensor preparation and all the experimental results suggesting that the proposed electrode could be used for electrocatalytic H2O2 detection in real samples.
Acknowledgement
This re s ea rc h was suppor te d by the Next - generation Medical Device Development Program for Newly-Created Market of the National Research F o u n d a t i o n ( N R F ) f u n d e d b y t h e K o r e a n government, MSIP (NRF-2015M3D5A1065760) to WS. HDJ is thankful to Science and Engineering Research Board (SERB), Government of India for the startup research grant (file no. SB/FT/CS-037- 2014).
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